DESCRIPTION: HIV specific CD8+ T cells, a pivotal arm of the antiviral immune response, exhibit progressive dysfunction, known as exhaustion. Severity of immune exhaustion correlates with disease progression, and is especially prevalent among drug abuse patients who often delay treatments that control HIV replication. PD-1, an immune inhibitory receptor and mediator of exhaustion in the setting of chronic infections and cancer, is upregulated on exhausted HIV-specific CD8+ T cells and in vitro blockade of its ligand with anti-PD- L1 antibodies restores, albeit partially, their immune functions. Given that exhausted T cells express multiple immune inhibitory receptors, the synergism of co-blockade of two receptors, and the limited effect of PD-L1 antibodies in vitro, strongly argues that PD-1 blockade alone will not be sufficient to rescue the full functionality of exhausted HIV specific CD8+ T cells. With the aim of developing a clinically feasible, cost-effective, and broadly applicable, treatment to reverse immune dysfunction in HIV-infected patients, our goal is to develop a single, chemically synthesized, nucleic acid based agent that can block the action of multiple inhibitory receptors in the same cell, and can be efficiently and specifically delivered to the exhausted T cells of the patient. The proposed approach is to inhibit downstream mediators of exhaustion like Blimp-1, Eomes, VHL or Sprouty-2, using RNAi in the form of siRNAs that are targeted to exhausted HIV-specific CD8+ T cells by conjugation to an oligonucleotide aptamer ligand that binds to receptors expressed on exhausted T cells, like PD-1, LAG3, orTim-3. The chemically synthesized aptamers, a novel platform for ligands with engineered specificity, offer significant advantages over the monoclonal antibody platform in terms of manufacture, cost, and reduced immunogenicity. Our lab has pioneered the use of aptamers as therapeutic agents and targeting ligands to modulate immunity in the setting of cancer, and have recently demonstrated the remarkable efficiency of aptamer-targeted siRNA delivery to circulating CD8+ T cells in mice. The central hypothesis of this proposal is that PD-1, LAG3, or Tim-3 aptamer targeted siRNA inhibition of mediators of exhaustion like Blimp- 1, Eomes, VHL or Sprout-2 will reverse the dysfunction of HIV-specific CD8+ T cells to an extent that is superior to that of blockade with monoclonal antibodies targeting inhibitory receptors like PD-1. The research plan is to generate high affinity aptamers to the aforementioned inhibitory receptors, construct aptamer-siRNA conjugates, and assess their ability, in comparison to blockade with antibodies, to restore immune functions in (i) Exhausted T cell cultured from HIV infected patients, (ii) HIV infected mice that are fully reconstituted with a human immune system, and (iii) an SIV/macaque model. This program will have developed a cost-effective and broadly applicable lead compound to test in patients chronically infected with HIV.